Regulation of mRNA 5'-3' interactions: The role of Paips

mRNA 5-3 相互作用的调节：Paips 的作用

• 批准号: 6941726
• 负责人: NAHUM SONENBERG
• 金额: $ 18.9万
• 依托单位: MCGILL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6941726
• 关键词: Drosophilidae; SDS polyacrylamide gel electrophoresis; binding proteins; biological signal transduction; electrofocusing; enzyme inhibitors; gene targeting; genetic recombination; genetic transcription; genetic translation; genetically modified animals; laboratory mouse; messenger RNA; phosphorylation; protein kinase; protein protein interaction; protein structure function; transposon /insertion element

DESCRIPTION (provided by applicant): The long-term objective of our research is to understand the biological function of a family of transaltional modulators known as PABP interacting protiens (Paips) which were first cloned in our laboratory. These proteins mediate their effects on translation by interacting with the poly(A) binding protein (PABP). Previous work has shown that Paipi stimulates translation, whereas Paip2A and Paip2B inhibit translation. The specific aims of this proposal are to further our understanding of the molecular mechanism of Paip action, to discover how Paip function is regulated, and to elucidate the biological significance of this family of proteins. A variety of in vitro and in vivo experiments will be carried out to study the Paip1PABP interactions, to dissect their mechanism of action, and to identify novel Paip interacting proteins. Paip expression and function will be inhibited by the RNA interference technique and the use of small cell-permeable peptides. Paip phosphorylation will be explored to elucidate the signaling pathways impinging upon Paip function. To this end, phosphopeptide mapping and 2-dimensional isoelectric focusing and SDS-PAGE will be used to study the phosphorylation states of the Paips under various environmental conditions and upon treatment with pharmacological kinase inhibitors. The phosphoresidues in the proteins will be identified and mutated. Resulting proteins will be assayed using in vitro and in vivo translation experiments to discover their functional importance. Elucidation of the physiological role of the Paips will be pursued by Paip overexpression and knockout (KO) experiments in Drosophila. Paip null flies will be generated by P-element insertion or homologous recombination. The generation of KO mice devoid of the individual Paip genes will also be pursued by homologous recombination. In the case of Paip2A and Paip2B, which are functional homologs, a Paip2AIPaip2B KO mouse will be generated. KO mice will be analyzed for phenotypic abnormalities.

描述（由申请人提供）：我们研究的长期目标是了解被称为 PABP 相互作用蛋白 (Paips) 的转换调节剂家族的生物学功能，该家族首先在我们的实验室克隆。这些蛋白质通过与聚腺苷酸结合蛋白 (PABP) 相互作用来介导其对翻译的影响。先前的研究表明 Paipi 刺激翻译，而 Paip2A 和 Paip2B 抑制翻译。该提案的具体目的是进一步了解Paip作用的分子机制，发现Paip功能是如何调节的，并阐明该蛋白家族的生物学意义。将进行各种体外和体内实验来研究 Paip1PABP 相互作用，剖析其作用机制，并鉴定新型 Paip 相互作用蛋白。 RNA 干扰技术和小细胞渗透性肽的使用将抑制 Paip 的表达和功能。将探索 Paip 磷酸化以阐明影响 Paip 功能的信号通路。为此，将使用磷酸肽图谱、二维等电聚焦和 SDS-PAGE 来研究 Paips 在各种环境条件下以及药理学激酶抑制剂治疗后的磷酸化状态。蛋白质中的磷酸残基将被识别并突变。将使用体外和体内翻译实验对所得蛋白质进行分析，以发现其功能重要性。 Paip 的生理作用将通过果蝇中的 Paip 过表达和敲除 (KO) 实验来阐明。 Paip null 果蝇将通过 P 元件插入或同源重组产生。缺乏单个 Paip 基因的 KO 小鼠的产生也将通过同源重组进行。对于功能同系物Paip2A和Paip2B，将生成Paip2AIPaip2B KO小鼠。将分析 KO 小鼠的表型异常。